Seroprevalence of Neospora caninum in slaughtered native cattle in Kurdistan province, Iran.

Neospora caninum is a worldwide distributed pathogen which causes abortion in cattle leading to economic loss in the cattle industry. The aim of this study was to determine the seroprevalence of N. caninum antibodies in the native cattle slaughtered in various areas of Kurdistan province (western Iran) from September 2010 to September 2011. Serum samples from 368 cattle slaughtered in seven slaughterhouses in this region were taken for detection of anti-N. caninum antibodies using commercial N. caninum ELISA kit. Antibodies to N. caninum were found in 29 samples (7.80%). The present study was the first report of Neospora infection in this region and indicated that native cattle of Kurdistan province were exposed to this parasite.


Introduction
Neospora caninum is a heteroxenous cyst-forming apicomplexan protozoan which is considered as a major causative of infectious bovine abortion worldwide and has been associated with sporadic, endemic and epidemic abortions. [1][2][3][4] The infection causes important economic loss to the cattle industry due to reproductive failure associated with abortion and mortality in congenitally infected calves. Neospora caninum infection has been reported in dairy cattle herds on all continents. 2,5 Dogs and coyotes are the definitive hosts in N. caninum life cycle, [6][7][8] whereas cattle and other mammals act as natural intermediate hosts. 9,10 In cattle, N. caninum infection may occurs by horizontal transmission due to ingestion of sporulated oocysts shed by the definitive host. 11,12 However, vertical transmission is the predominant route of infection. 1,12 Vertical transmission occurs when tachyzoites cross the placenta of a persistently infected dam and infect the fetus. 13 Transplacental transmission can occur in consecutive pregnancies in the same cattle and so the infection can persist in cattle herds through many generations. 14,15 The infection usually has a chronic course and persists throughout the life of an infected animal. 16 Neospora caninum DNA has been reported in fresh and frozen semen of naturally infected bulls and the possibility of venereal transmission in bovine neosporosis has been suggested. [17][18][19] Non-pregnant adult cattle infected with N. caninum do not show any signs of disease, but infection in a pregnant animal may cause abortion, or in the birth of weak, diseased sub-clinically infected or healthy calves. 2,20 Abortion may occur at any stage of pregnancy irrespective of whether the infection in the cow is recent, chronic or congenital. 2,21 Since recognition of N. caninum in 1980, there are only few studies about the seroprevalence of bovine neosporosis in Iran, which are mostly carried out in Tehran 21 , Mashhad north eastern of Iran, 22,23 and Kerman south eastern of Iran. 24 Although there were some reported about seroprevalence of N. caninum in industrial cattle in Iran, [21][22][23][24] there was no published studies in native cattle. As there was not any published study about seroprevalence of N. caninum in native cattle of west part of Iran, this study was performed to determine the prevalence of antibodies to N. caninum in native cattle in Kurdistan province, Iran.

Materials and Methods
Study area. This study was carried out in seven different slaughterhouses of Kurdistan province, western Iran. Kurdistan has an area of about 28,203 Km 2 . According to Iranian Veterinary Organization in 2009, an average of 321,557 native cattle exist in this area.
Sampling. Blood samples, were taken randomly from 368 female native cattle of three main geographical zones including seven slaughterhouses of western, central and eastern regions of this province from September 2010 to September 2011. Age of animals was determined according to dental formula and then was matched with owners' information. All samples were immediately transferred to laboratory of parasitology (Faculty of Veterinary Medicine, Bu-Ali Sina University, Hamedan, Iran). The cattle were divided into four age groups (< 2 years, 2-6 years, 6-8 years and > 8 years old). Blood samples were centrifuged at 1000 g for 10 min and then serum was carefully removed. All sera were stored at -20 ˚C until laboratory testing.
Sera were tested for the presence of anti-N. caninum antibodies using ELISA Kit (Herdcheck, Maine, USA). According to the manufacturer's instruction, the presence or absence of antibodies to N. caninum was determined by sample to positive (S/P) ratio for each sample. Serum samples with S/P ratios less than 0.50 were classified as negative and greater than or equal to 0.50 were classified as positive. All data were analyzed by Chi-square test using SPSS (Version 9.0; SPSS Inc., Chicago, USA). A p-value of less than 0.05 was considered statistically significant.

Results
Anti-N. caninum antibodies were detected in 29 of 368 cattle sera (7.80%). The rate of seropositive slaughtered animals in seven different slaughterhouses are presented in Table 1. The difference between prevalence of anti-N. caninum antibodies in slaughtered animals in different slaughterhouses was significant (χ 2 = 17.492, df = 6, p < 0.05). The seroprevalence of anti-N. caninum antibodies in different age group are shown in Table 1. There was not an age dependent response as χ 2 = 0.620, df = 3, p = 0.895. There was no significant difference in the prevalence of abortion between seropositive and non-seropositive cattle (χ 2 = 0.329, df = 1, p = 0.588) indicating that N. caninum was not an important causative agent of abortion in the cattle slaughtered in seven different mentioned slaughterhouses in Kurdistan province (Table 2).

Discussion
Neospora caninum is considered to be one of the major causative agent of abortion in cattle worldwide. 25 Several serologic tests including ELISA, immunofluorescent antibody test (IFAT), and direct agglutination test (DAT) were employed to detect anti-N. caninum antibodies. 2,24 At the present, the most commonly serological tests used for the diagnosis of Neospora infection are IFAT and ELISA. 24 The present study showed that the seroprevalence of N. caninum infection in the native cattle of Kurdistan province is 7.80%. Our results were nearly similar with the results of investigation on cattle neosporosis of Kars province in Turkey (8.20%). 26 Antibodies level would be expected to be at its peak level if N. caninum were involved in abortion. 27 Results of the previous studies were in close agreement with findings of the present study because there was no significant difference in the prevalence rate of abortion between seropositive and non-seropositive cattle (p = 0.588). [26][27] The present study suggested that N. caninum was not an important causative agent of abortion in local cattle of Kurdistan province. This finding maybe due to that native breeds are genetically resistant to neosporosis. 2 In investigation in Brazil on the infection of N. caninum in different cattle breeds (Zebu, Holstein, crossbreed Zebu/ Holstein) there was close association between cattle breeds and the frequency of infection by N. caninum. The result of this study indicated that breed, which is inherent factor to host, could be considered of high relevance for the distribution and frequency of infection by N. caninum. The authors of this study suggested that genetic distance between Holstein and Zebu breeds possibly leads to the significant difference. 28 These results were similar to finding of a study performed in Ontario which showed that there was a genetic susceptibility to infection by N. caninum in Holstein cattle. 29 Few studies have been carried out considering breed as an epidemiological parameter, to understand the distribution of the infection by N. caninum in cattle, probably due to the fact that most of the surveys are restricted to pure breed cattle or to very similar breeds. 29 In the present study, comparison of N. caninum serological status with age groups showed no significant difference (p = 0.895). This finding was in close agreement with the result of other studies. 19,22 The authors of these studies reported that for most herds seroprevalence levels were equal across all age groups.
In conclusion, the overall seroprevalence of N. caninum in native cattle of Kurdistan province was low, suggesting that N. caninum was not an important causative agent of abortion in the animals studied in the region. However, further studies on the epidemiological aspect of N. caninum infection in cattle and the relationship between abortion in cattle and infection with N. caninum in native cattle of this region are required.